BpCYP76AD15 is involved in betaxanthin biosynthesis in bougainvillea callus

Main conclusion BpCYP76AD15 is involved in betaxanthin biosynthesis in callus, but not in bracts, in bougainvillea. Bougainvillea ( Bougainvillea peruviana ) is a climbing tropical ornamental tree belonging to Nyctaginaceae. Pigments that are conferring colorful bracts in bougainvillea are betalains, and that conferring yellow color are betaxanthins. In general, for red-to-purple betacyanin biosynthesis, α clade CYP76AD that has tyrosine hydroxylase and DOPA oxygenase activity is required, while for betaxanthin biosynthesis, β clade CYP76AD that has only tyrosine hydroxylase is required. To date, betaxanthin biosynthesis pathway genes have not been identified yet in bougainvillea. Since bougainvillea is phylogenetically close to four-O-clock ( Mirabilis jalapa ), and it was reported that β clade CYP76AD, MjCYP76AD15 , is involved in floral betaxanthin biosynthesis in four-O-clock. Thus, we hypothesized that orthologous gene of MjCYP76AD15 in bougainvillea might be involved in bract betaxanthin biosynthesis. To test the hypothesis, we attempted to identify β clade CYP76AD gene from yellow bracts by RNA-seq; however, we could not. Instead, we found that callus accumulated betaxanthin and that β clade CYP76AD gene, BpCYP76AD15 , were expressed in callus. We validated BpCYP76AD15 function by transgenic approach (agro-infiltration and over-expression in transgenic tobacco), and it was suggested that BpCYP76AD15 is involved in betaxanthin biosynthesis in callus, but not in bracts in bougainvillea. Interestingly, our data also indicate the existence of two pathways for betaxanthin biosynthesis (β clade CYP76AD-dependent and -independent), and the latter pathway is important for betaxanthin biosynthesis in bougainvillea bracts.